I. Retroviruses
The Retroviridae virus family encompasses all viruses containing an RNA genome and producing an RNA-dependent DNA polymerase (reverse transcriptase). In broadest overview, the life cycle of a retrovirus comprises entry of an infectious retroviral particle into a host cell, integration of the virus' genetic information into the host cell's genome, and production of new infectious retroviral particles by the biosynthetic machinery of the infected host cell. More specifically, upon entering a cell, a retroviral particle initiates a series of interactive biochemical steps that result in the production of a DNA copy of the virus' RNA genome and its integration into the nuclear DNA of the cell. This integrated DNA copy is referred to as a provirus and can be inherited by any daughter cells of the infected cell like any other gene. Genes contained within the integrated provirus may be expressed in the host cell.
All retroviral particles share common morphological, biochemical, and physical properties, including:
(1) A linear, positive-sense, single-stranded RNA genome composed of two identical subunits and making up about 1% of the mass of the virus. PA0 (2) At least three types of proteins encoded by the viral genome, i.e., gag proteins (the group antigen internal structural proteins), pol proteins (the RNA-dependent DNA polymerase and integrase proteins), and env proteins (the viral envelope protein or proteins). These proteins together make up about 60%-70% of the mass of the virus. PA0 (3) Lipid derived from the cell membrane of an infected cell making up about 30%-40% of the mass of the virus. PA0 (4) Carbohydrate associated with the env proteins, making up about 2-4% of the mass of the virus. PA0 (5) An overall spherical morphology with variable surface projections. PA0 (6) An isocahedral capsid structure containing a ribonucleoprotein complex within an internal nucleoid or nucleocapsid shell. PA0 (1) Gal .alpha.(1,3) Gal .beta.(1,4) GlcNAc, PA0 (2) Gal .alpha.(1,3) Gal .beta.(1,4) Glc, PA0 (3) Gal .alpha.(1,3) Gal .beta., and PA0 (4) Gal .alpha.(1,3) Gal.
In addition to genes encoding the gag, pol, and env proteins, the genome of the retrovirus includes two long terminal repeat (LTR) sequences, one at each end of the linear genome. These 5' and 3' LTRs serve to promote transcription and polyadenylation of viral mRNAs. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the viral genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site). Other genes may also be found between the 5' and 3' LTRs of the retroviral genome.
If heterologous genes are inserted in between the 5' and 3' LTRs of a retroviral genome, which is then packaged into a functional retroviral particle, the resulting recombinant retroviral particle is capable of carrying the heterologous genes into a host cell. Upon integration of the recombinant retroviral genome into the host cell's genome as part of the proviral DNA, the heterologous genes may be expressed.
These properties and capabilities have led to the development of retroviral vectors, retroviral packaging and producer cells, which are typically prepared from cells of murine origin, and retroviral vector particles (collectively referred to as retroviral transduction systems) as efficient means of stably introducing exogenous genes of interest into mammalian cells. Certain retroviruses have been engineered to produce non-infectious retroviral transduction systems that are especially useful in the field of gene therapy. See, for example, Anderson, 1992; Miller, 1992; Mulligan, 1983; Mann, 1983; Cone and Mulligan, 1984.
II. Gene Transfer by Retroviral Transduction
Retroviral transduction systems of the type discussed above are able to introduce recombinant nucleic acid molecules into mammalian target cells, and to efficiently integrate DNA molecules containing some or all of the genetic information (sequence) of the introduced recombinant nucleic acid molecule into the genome of the target cell so that the introduced genetic material is replicated and is stably and functionally maintained (and any encoded gene products are expressed) in the cell without the danger of the production of replicating infectious virus. See, for example, Ausubel, et al., Volume 1, Section III (units 9.10.1-9.14.3), 1992.
Retroviral vector particles are particularly useful for genetically modifying mammalian cells, including human cells, because the efficiency with which they can transduce target cells and integrate their genetic information into the target cell genome is higher than that achievable using other systems of introducing exogenous genetic material into cells. Other advantages associated with the use of retroviral vector particles as gene therapy agents include stable expression of transferred genes, capacity to transfer large genes, and lack of cellular cytotoxicity. Additionally, retroviral vector articles may be constructed so as to be capable of transducing mammalian cells from a wide variety of species and tissues.
Successful gene transfer by transduction with a retroviral vector particle (RVVP) requires: 1) incorporation of a gene of interest into a retroviral vector; 2) packaging of a vector-derived viral genome into a RVVP; 3) binding of the RVVP to the target cell; 4) penetration of at least the RNA molecules comprising the viral genome into the target cell (generally associated with penetration of the RVVP and uncoating of the RVVP); 5) reverse transcription of the viral RNA into pre-proviral cDNA; 6) incorporation of the pre-proviral cDNA into preintegration complexes, 7) translocation of the preintegration complexes into the target cell nucleus, 8) generation of stable proviral DNA by integration of the pre-proviral cDNA into the host genome (typically mediated by the viral integrase protein); and 9) expression of the gene of interest. In the in vivo setting (and in some ex vivo settings), the RVVP must survive in the extracellular fluids of the host organism in an active state for a period sufficient to allow binding and penetration of the host target cell by the RVVP.
Gene Therapy: There is active research, including clinical trial research, on treatment of disease by introduction of genetic material into some of the cells of a patient. A variety of diseases may be treated by therapeutic approaches that involve stably introducing a gene into a cell such that the gene may be transcribed and the gene product may be produced in the cell. Diseases amenable to treatment by this approach include inherited diseases, particularly those diseases that are caused by a single gene defect. Many other types of diseases, including acquired diseases, may also be amenable to gene therapy. Examples of such acquired diseases include many forms of cancer, lung disease, liver disease, and blood cell disorders. See Anderson, 1992; Miller, 1992; and Mulligan, 1993.
Delivery of the gene or genetic material into the cell is the first critical step in gene therapy treatment of disease. A variety of methods have been used experimentally to deliver genetic material into cells. Most research has focused on the use of retroviral and adenoviral vectors for gene delivery. As discussed above, RVVPs are particularly attractive because they have the ability to stably integrate transferred gene sequences into the chromosomal DNA of the target cell and are very efficient in stably transducing a high percentage of target cells. Accordingly most clinical protocols for gene therapy use retroviral vectors (see, for example, Miller, 1992; and Anderson, 1992).
Most gene therapy protocols involve treating target cells from the patient ex vivo and then reintroducing the cells into the patient. Patients suffering from several inherited diseases that are each caused by a single gene defect have already received gene therapy treatments. Such treatments generally involve the transduction of the patient's cells in vitro using RVVPs designed to direct the expression of therapeutic molecules, followed by reintroduction of the transduced cells into the patient. In many cases such treatments have provided beneficial therapeutic effects.
For many diseases, however, it will be necessary to introduce the gene into the target cell in situ, because the target cells cannot be removed from and returned to the body. In other cases, cells that are removed from the patient must be maintained in the presence of body fluids until being returned to the body. Stem cells, particularly hematopoietic stem cells, are an especially important type of target cell for gene therapy of inheritable and acquired blood disorders. Such cells are intrinsically unstable in vitro, and tend to differentiate into cells that are less attractive targets for gene therapy, especially when they have been washed free of the fluids that surround them in vivo and transferred into body-fluid-free tissue culture media or the like.
Accordingly, it is desirable to transduce stem cells as quickly as possible, and ex vivo treatment of such cells with RVVPs is best carried out in the cells natural milieu, i.e., in cells that have not been washed or otherwise removed from the body fluids in which they are obtained, e.g., hematopoietic stem cells in bone marrow aspirates. In the case of stem cells in bone marrow, current medical procedures for bone marrow transplant involve mixing an ex vivo bone marrow aspirate (which is inevitably obtained as a mixture of bone marrow and blood) with an anticoagulant, typically heparin, and tissue culture medium. The condition of such cells, that have been removed from the body but kept in diluted or undiluted fluids of their natural milieu, is referred to hereinafter as the "ex vivo unwashed state".
III. The Humoral Immune System and Retroviral Vector Particles
A longstanding problem associated with the use of RVVPs as gene therapy vectors in cells in vivo or in cells in the ex vivo unwashed state relates to the inactivation of many retroviruses (and RVVPs derived therefrom) by the body fluids (e.g., blood, bone marrow, lymph) of many primates, including Old World monkeys, apes, and humans. Indeed, it has been known for almost two decades that certain retroviruses are rapidly inactivated in human serum (Welsh et al., 1975), as well as serum from nonhuman primates (Welsh et al., 1976). This problem has precluded the use of such RVVPs for gene therapy in vivo or in the ex vivo unwashed state.
The humoral immune system, and particularly the complement system has long been implicated in the serum mediated inactivation of retroviruses, as serum deficient in C2, C4 or C8 does not cause the detectable release of reverse transcriptase from retroviral virions (Welsh et al., 1975; Cooper, et al., 1976). The protection of active retroviral particles from human complement is thus necessary for the use of the RVVPs to mediate gene therapy in human cells in vivo or in the ex vivo unwashed state. Accordingly, to date, gene transfer by retroviral transduction has been, for the most part, limited to cells that were removed from the extracellular fluids of the host organism (i.e., ex vivo cells that are not in the ex vivo unwashed state) and thus were not subjected to complement attack. This limitation has represented a significant shortcoming of this technology.
The need for methods allowing transduction of primate cells in situ, in vivo, or in the ex vivo unwashed state has resulted in the development of methods designed to prevent the inactivation of retroviruses by human and other primate sera. Such methods have included the removal of cells from the extracellular fluids of the host organism, as discussed above, as well as the masking of virion structures that can activate complement activity by administration of isolated C1s and/or C1q complement subcomponents, as discussed below under the subheading "The Direct C1 Binding Mechanism".
Significantly, with regard to the present invention, no previous methods for allowing transduction of primate cells in situ, in vivo, or in the ex vivo unwashed state have included methods or compositions for preventing antibody binding to alpha galacotsyl epitopes on virion cell surface molecules.
The Direct C1 Binding Mechanism: Retroviruses that are sensitive to human serum have been reported to activate the human classical complement pathway by a mechanism that involves an antibody independent process. This process is found in many primates and is generally not present in other mammals (see Cooper, et al., 1976). This mechanism is activated when complement component C1 binds to retroviral virions directly and triggers the classical complement pathway, just as the pathway is normally activated by an antigen-antibody complex (Bartholomew, et al., 1978). The complement cascade then causes the eventual destruction and elimination of the virus. Prior to the present invention, it has generally been believed in the art that this mechanism provides the major, if not the only, means by which retroviral virions are destroyed by the humoral immune system.
Complement component C1 is a large complex protein composed of 3 subunits designated C1q, C1s, and Clr. C1q is itself composed of 18 polypeptide chains of three different types designated A, B, and C. Six molecules each of chains A, B, and C compose the C1q subunit. There are two molecules each of the C1s subunit and the Clr subunit that associate with C1q to form the C1 complement component. The C1q subunit contains multiple identical binding sites for the complement binding regions of immunoglobulin molecules, which regions are only exposed upon the formation of an antigen-antibody complex. In the classical pathway, the binding of C1q to these regions of antigen-bound antibody molecules causes a conformational change in the Cl complex resulting in the enzymatic activation of C1 to yield an active serine protease. The C1s and C1q subunits both have a molecular weight of approximately 85 kDa, and each is cleaved to smaller molecular weight forms of approximately 57 kDa and 28 kDa during activation of the C1 complex. The 57 kDa forms of C1s and C1q present in the activated C1 complex contain the protease activity.
In the activation of the classical complement pathway by retroviruses via direct binding of C1, the C1q subunit of Cl binds directly to at least one site on the retroviral virion. In the case of Moloney murine leukemia virus, the pl5E viral protein has been identified as the C1 binding receptor. See Bartholomew, et al., 1978. In contrast to the antibody-mediated classical complement pathway, binding by both the C1q subunit and the C1s subunit of the Cl complex is required for complement activation by retroviral particles via this mechanism. Furthermore, the C1s subunit and C1q subunit must bind the viral particle when they are present in a functional Cl complex in order for complement activation to occur by this mechanism. See Bartholomew, et al., 1980.
The C1s subunit is also believed to have a specific binding site for retroviral coat proteins. It has been shown, using inactive retrovirus, that pre-binding with C1s blocks the subsequent activation of the complement cascade by the retrovirus in vitro. See Bartholomew, et al., 1980.
Co-pending U.S. patent application Ser. No. 08/098,944 ("the '944 application"), filed Jul. 28, 1993 in the name of James M. Mason and entitled "Pre-binding of Retroviral Vector Particles with Complement Components to Enable The Performance of Human Gene Therapy In Vivo," discuses the use of free C1q or free C1s to block the subsequent binding and/or activation of the C1 complex by active retrovirus particles including RVVPs.
As described therein, C1s or C1q or a combination thereof are incubated with the RVVPs in vitro to form complexes with the particles. This complex formation blocks the binding sites for C1s and/or C1q and thereby protects the particles from subsequent inactivation or lysis when the RVVPs are exposed to complement. As further described therein, blockade of intact C1 binding to RVVPs can be achieved by the use of fragments of antibodies that bind the viral envelope proteins of RVVPs but lack complement binding regions.
As disclosed in the '944 application, the use of these methods improves the survival of RVVPs in human serum, but does not completely inhibit retroviral inactivation. The incomplete nature of the inhibition of retroviral inactivation by these methods have heretofore been unexplained.
Many gene therapy methods require very high titers of transducing RVVPs in order to be practiced effectively. The methods of the '944 application are of limited efficacy, as they do not provide a sizable inhibition of complement mediated RVVP inactivation, and thus may not provide high enough titers of RVVPs in vivo or in the ex vivo unwashed state for the effective practice of all such gene therapy methods.
Thus, a need continues to exist for methods to control complement-mediated destruction of RVVPs. The present invention provides new methods and compositions that can be used in conjunction with, or as an alternative to other methods of protecting RVVPs from inactivation by complement such as the blockade of intact C1 binding to RVVPs as disclosed in the '944 application. The methods and compositions of the present invention thus allow the practice of more efficient gene therapy procedures in vivo, in situ, and in the ex vivo unwashed state.
IV. The Complement System
The complement system acts in conjunction with other immunological systems of the body to defend against intrusion of cellular and viral pathogens. There are at least 25 complement proteins, which are found as a complex collection of plasma proteins and membrane cofactors. The plasma proteins (which are also found in most other body fluids, such as lymph, bone marrow, and cerebrospinal fluid) make up about 10% of the globulins in vertebrate serum. Complement components achieve their immune defensive functions by interacting in a series of intricate but precise enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory, and lytic functions.
The complement cascade progresses via the classical pathway or the alternative pathway. These pathways share many components, and, while they differ in their early steps, both converge and share the same terminal complement components responsible for the damage and destruction of target cells and viruses.
The classical complement pathway is typically initiated by antibody recognition of and binding to an antigenic site on a target cell. This surface bound antibody subsequently reacts with the first component of complement, C1, which, as discussed above, includes subunits C1s, Clr, and C1q.
The C1q subunit of C1 mediates the binding of C1 both to antigen-antibody complexes and to retroviruses, although, in the case of direct binding to retroviruses, the C1s subunit also has a binding function. The bound C1 undergoes a set of autocatalytic reactions that result in the activation of the Clr subunits, which in turn proteolytically activate the C1s subunits, altering the conformation of C1 so that the active C1s subunits are exposed on the exterior of C1, where they can interact proteolytically with complement components C2 and C4.
C1s cleaves C2 and C4 into C2a, C2b, C4a, and C4b. The function of C2b is poorly understood. C2a and C4b combine to form the C4b,2a complex, which is an active protease known as the C3 convertase. C4b,2a acts to cleave C3 into C3a and C3b. C3a is a relatively weak anaphylatoxin. C4a is a stronger anaphylatoxin, and can induce degranulation of mast cells, resulting in the release of histamine and other mediators of inflammation.
C3b has multiple functions. As opsonin, it binds to bacteria, viruses and other cells and particles and tags them for removal from the circulation. C3b can also form a complex with C4b,C2a to produce C4b,2a,3b, or C5 convertase, which cleaves C5 into C5a (another anaphylatoxin), and C5b. C5b combines with C6 yielding C5b,6, and this complex combines with C7 to form the ternary complex C5b,6,7. The C5b,6,7 complex binds C8 at the surface of a cell membrane. Upon binding of C9, the complete membrane attack complex (MAC) is formed (C5b-9) which mediates the damage and lysis of foreign cells, microorganisms, and viruses.
A more complete discussion of the classical complement pathway, as well as a detailed description of the alternative pathway of complement activation, which pathway has also been implicated in the inactivation of RVVPs by human complement, can be found in Roitt, et al., 1988.
V. Galactose Alpha (1,3) Galactosyl Epitopes
Natural human antibodies are preformed antibodies that bind to epitopes of foreign antigens (xenoepitopes). Several recent studies have convincingly demonstrated that the galactose alpha (1,3) galactosyl carbohydrate epitopes, also referred to as Gal .alpha.(1,3) Gal epitopes, are major xenoepitopes recognized by natural human antibodies (see Sandrin, et al., 1993A; Sandrin, et al., 1993B; copending U.S. patent application Ser. No. 08/214,580, entitled "Xenotransplantation Therapies", filed by Mauro S. Sandrin and Ian F. C. McKenzie on Mar. 15, 1994; copending U.S. patent application Ser. No. 08/278,282, entitled "Methods for Reducing Hyperacute Rejection of Xenografts", filed Jul. 21, 1994 in the names of Mauro S. Sandrin, William L. Fodor, Russell P. Rother, Stephen P. Squinto, and Ian F. C. McKenzie; and PCT publication No. 93/03735, entitled "Methods and Compositions for Attenuating Antibody-Mediated Xenograft Rejection"). In addition, it has been suggested that galactose alpha (1,3) galactosyl epitopes on certain DNA viruses may be involved in triggering immune responses (Repik et al., 1994).
Galili and colleagues have shown that a large proportion of IgG (1%) in human serum is directed against the galactose alpha (1,3) galactosyl epitopes expressed as part of a variety of glycosylated molecules found on both cell surfaces and on secreted glycoproteins (Galili et al., 1984; and Thall and Galili, 1990). This disaccharide epitope is found in all mammals except humans and Old World primates, and naturally occurring preformed anti-.alpha.(galactosyl antibodies e.g., anti-galactose alpha (1,3) galactose antibodies--i.e., antibodies that bind specifically to galactose alpha (1,3) galactosyl epitopes--are found only in humans and Old World primates, i.e., those species that do not themselves express the epitope (Galili et al., 1987 and Galili et al., 1988).
The ability of different monosaccharides and oligosaccharides to inhibit the interaction of naturally occurring preformed human antibodies with pig cells and to prevent the antibody-dependent and complement-mediated damage and lysis of pig cells has been examined (Sandrin et al., 1993A; Sandrin et al., 1993B; PCT publication No. 93/03735, supra; and copending U.S. patent application Ser. No. 08/214,580, supra).
Inhibition of the binding of such antibodies to xenogeneic cells was obtained with galactose, or with moieties containing terminal galactose in an alpha linkage but not a beta linkage. Various carbohydrates have also been shown to contain the target epitopes for several types of naturally occurring preformed human antibodies with other specificities (e.g., ABO blood group antibodies). However, no monosaccharide tested, other than those containing the galactose alpha (1,3) galatosyl epitope, had any inhibitory effect on the binding of naturally occurring preformed human antibodies to xenogeneic cells. Identical inhibition results were obtained when individual human serum samples from blood group A, B, AB or O individuals were used (Sandrin et al., 1993A and Sandrin et al., 1993B).
Similarly, Cooper and colleagues have demonstrated that, of a total of 132 carbohydrates screened for binding to preformed naturally occurring human IgG and IgM antibodies, each of the four carbohydrate molecules that they found could bind such antibodies contained a terminal alpha galactose (Good et al., 1992). The four carbohydrates were:
Sugars such as melibiose (a disaccharide containing a terminal galactose in an alpha (1,3) linkage) coupled to a carrier such as SEPHAROSE can be used to purify anti-galactose alpha (1,3) galactose antibodies (Galili et al, 1984 and Galili et al., 1985). In some antibody absorption experiments, human serum was passed over the carrier-sugar matrix in order to prepare serum from which the antibodies reactive with the sugar were removed. The results of testing the cytolytic activity of the sera prepared in these experiments indicate that the majority of the cytotoxic antibodies were removed from the serum by these means (Sandrin et al., 1993A; Sandrin et al., 1993B).
In sum, the results of the sugar inhibition studies, the studies of the binding of antibodies to galactose alpha (1,3) galatosyl epitope-containing molecules, and the studies of the absorption of antibodies by melibiose-SEPHAROSE, all lead to the conclusion that galactose alpha (1,3) galactosyl epitopes are amongst the most important epitopes detected by naturally occurring human antibodies.
Inhibitors of Glycosylation
Glycosylation of retroviral proteins, including the glycosylated envelope protein (gp70), is a dynamic process involving the host cell translational machinery. Mature glycoproteins which contain asparagine linked (N-linked) oligosaccharides fall generally into three categories, depending on the oligosaccharide side chains of their carbohydrate moieties: high mannose, complex, and hybrid types. These side chain oligosaccharides are added to nascent proteins through a well characterized biosynthetic pathway (for review see Kornfeld and Kornfeld, 1985).
This pathway is initiated with the addition of a glucosylated high mannose oligosaccharide precursor, (Glc).sub.3 (Man).sub.9 (GlcNAc).sub.2. This high mannose precursor is trimmed by various glucosidases and mannosidases as the protein traverses the rough endoplasmic reticulum and golgi apparatus, respectively. When high mannose oligosaccharide side chains are not trimmed by the mannosidases, the high mannose type side chain results. When high mannose oligosaccharide sides chains are trimmed by the mannosidases and are subsequently modified to contain glucosamine, fucosyl, galactosyl, and other side chain additions, the complex type side chain results. Intermediates between these two end products are termed hybrid side chains.